This invention relates to a process for selectively removing protein aggregates from a protein solution. More particularly, this is invention relates to a process for selectively removing protein aggregates and virus from a protein solution.
Protein solutions such as immunoglobulin protein (IgG) including polyclonal antibodies routinely contain protein aggregates comprising protein trimers or higher polymers. In order to administer this solution to a patient, it is necessary to first remove these aggregates to avoid a toxic response by the patient. When utilizing conventional filtration processes, aggregates are undesirable since the filter rapidly becomes plugged by the aggregates even at low aggregate concentrations of 0.1-0.2%. Accordingly, it has been necessary to utilize expensive gel chromatography or size exclusion chromatography processes to effect selective aggregate removal.
Virus also are a potential contaminant in parenteral and other solutions containing a protein which are derived from either whole organisms or mammalian cell culture sources. Currently several chemical and physical methods exist to inactivate virus. These methods are not generic to all virus equally and some operate at the expense of protein activity. For example, heat pasteurization is used in solutions where protein denaturization can be minimized through the addition of stabilizers. In the biotechnology industry, strategies have been adopted that combine several inactivation or removal steps in the downstream process to maximize virus removal capability and protein recovery. The operations used are generally those operations optimized to purify the parenteral product and are validated for the virus removal capability. Thus, virus removal is an additional capability from a by-product of normal operation. Finally, at the end of the process, steps such as chromatography, filtration or heat may be added to increase overall virus clearance. This strategy has two shortcomings; (1) the virus clearance of these operations may not apply to putative virus that cannot be assayed; and (2) the virus clearance of the process needs to be monitored continually. It is necessary to remove virus at a log retention value at least 3, i.e., at least about 99.9% removal.
Accordingly, it would be desirable to provide a process for removing protein aggregates from a protein solution by a filtration process which avoids premature plugging of the filtration membrane utilized in the process. In addition, it would be desirable to provide such a process which can be utilized in conjunction with a process for removing virus from the protein solution at a log retention value of at least 3.
The present invention provides a process for removing protein aggregates comprising protein dimers, protein trimers and higher protein polymers from a protein solution. The protein solution containing the aggregates are filtered through a cellulosic ultrafiltration membrane having a molecular weight cutoff between about 500 kD and about 1000 kD (the molecular weight of the solute that is 90% rejected by the membrane under low polarization conditions). The feed is filtered by tangential flow filtration wherein the feed is passed tangentially across the membrane surface to produce a retentate stream and a permeate stream. Filtration is effected using a transmembrane pressure between about 1 and about 10 psi. The retentate is recycled to a reservoir for the protein solution feed under conditions of essentially constant protein concentration in the feed by adding a buffer solution to the retentate. When filtering a protein solution containing virus, the filter utilized can retain virus particles. The filtration membrane is periodically washed with water or aqueous buffer to remove retained protein aggregates thereby to permit reuse of the membrane.
When utilizing a second filtration step to selectively retain virus, filtration can be effected with an ultrafiltration membrane either by tangential flow filtration (TFF) or by dead end (normal) filtration (NFF) wherein a permeate stream is produced while avoiding the formation of a retentate stream. The ultrafiltration membrane retains virus particles while permitting passage of protein monomer therethrough. When utilizing TFF, the retentate stream is recycled to a reservoir and then into contact with membrane(s) until substantially all of the protein is passed through the membrane. Subsequent to the filtration step, the membrane can be flushed with water or an aqueous buffer solution to recover any protein retained by the membrane.
The use of the two step process of this invention to remove protein aggregates and virus particles from a protein solution provides substantial advantages over the one step filtration process of the prior art. Since the membrane used in the first step of removing aggregates is washed with water, buffer or a cleaning agent to remove the retained aggregate, the membrane can be reused for the same purpose of removing protein aggregates from a protein solution. In addition, since the membrane utilized in the second step of removing virus particles does not foul with protein aggregates, its useful life is extended since it does not become plugged with protein aggregates.